Methods targeting miR-128 for regulating cholesterol/lipid metabolism

ABSTRACT

Methods for targeting microRNA 128 (miR-128) for regulating cholesterol/lipid metabolism and insulin sensitivity.

CLAIM OF PRIORITY

This application is a U.S. National Phase Application under 35 U.S.C.371 of International Patent Application No. PCT/US2012/021257, filed onJan. 13, 2012, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/432,991, filed on Jan. 14, 2011. The entire contents of theforegoing are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grants No.R21DK084459 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

This invention relates to methods for targeting microRNA-128 (miR-128)for regulating cholesterol/lipid metabolism and insulin sensitivity,inter alia.

BACKGROUND

Abnormal cholesterol and lipid homeostasis are linked with prevalentdiseases such as metabolic syndrome, atherosclerosis/cardiovasculardisease, and type 2 diabetes. Cholesterol and lipids are trafficked inthe blood as lipoprotein particles, such as low-density lipoprotein(LDL) and high-density lipoprotein (HDL) that ferry their fatty cargo todifferent cells and tissues. Excess circulating LDL can be oxidized andtaken up by arterial macrophages, turning them intocholesterol/lipid-filled “foam cells” that are involved in the formationof atherosclerotic plaques. Triglycerides, as major components ofvery-low-density lipoprotein (VLDL), have been linked toatherosclerosis, and, by extension, the risk of heart disease andstroke. Elevated, triglycerides (e.g., mildly elevated fasting levels,above 150 mg/dL (1.7 mmol/L), or high fasting levels above 200 mg/dL(2.26 mmol/L)) are common in subjects with metabolic syndrome/insulinresistance and those with poorly controlled diabetes, and contribute tothe risk of atherosclerosis, heart disease, and stroke in thatpopulation.

SUMMARY

As shown herein, miR-128-1 targets a large number of genes/proteinsinvolved in cholesterol/lipid homeostasis and insulin signaling. Thedata presented herein demonstrates that low density lipoprotein receptor(LDLR), ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1),Sirtuin 1 (SIRT1), and Insulin Receptor Substrate 1 (IRS1) are regulatedby miR-128-1. Indeed, antisense targeting of miR-128-1 in human livercells (HepG2) results in increased expression of these keycholesterol/lipid regulators and insulin signaling proteins.Dys-regulation of these proteins in humans is thought to contribute toaberrant cholesterol/lipid homeostasis and insulin resistance inMetabolic Syndrome and cardiovascular disease patients. Thus, themethods described herein can be used to normalize cholesterol/lipidhomeostasis and decrease insulin resistance. For example, in someembodiments the methods described herein include the use of inhibitorynucleic acids to target miR-128-1 to improve cholesterol/lipidhomeostasis and insulin resistance, e.g., in subjects with MetabolicSyndrome and/or cardiovascular disease.

Thus, in a first aspect, the invention provides methods for reducinglevels of serum LDL, reducing levels of serum triglycerides, and/orincreasing levels of serum HDL in a subject. The methods includeadministering to the subject a therapeutically effective amount of aninhibitory nucleic acid that is complementary to all or part of any ofSEQ NOs:1-6, e.g., to all or part of SEQ ID NO:2.

In another aspect, the invention provides methods for treating orreducing the risk of developing diabetic neuropathy, non-alcoholic fattyliver disease, non-alcoholic steatohepatitis, atherosclerosis, and/orcardiovascular disease in a subject. The methods include administeringto the subject a therapeutically effective amount of an inhibitorynucleic acid that is complementary to all or part of any of SEQ NOs:1-6,e.g., to all or part of SEQ NO:2.

In an additional aspect, the invention provides methods for reducingobesity, treating or reducing predisposition to insulin resistance,and/or treating or reducing predisposition to type II diabetes, in asubject. The methods include administering to the subject an inhibitorynucleic acid sequence that is complementary to all or part of any of SEQNOs:1-6, e.g., to all or part of SEQ NO:2, thereby decreasing obesity inthe subject.

In a further aspect, the invention provides methods for increasinguptake of lipids or cholesterol by a cell, e.g., a liver cell, or forincreasing sensitivity of a cell, e.g., a liver cell, to insulin. Themethods include contacting the cell with an inhibitory nucleic acidsequence that is complementary to all or part of any of SEQ NOs:1-6,e.g., to all or part of SEQ ID NO:2.

In some embodiments of the methods described herein, the inhibitorynucleic acid is complementary to at least nucleotides 2-7 (5′-CACAGU-3′)of SEQ ID NO:3. In some embodiments of the methods described herein, theinhibitory nucleic acid can be designed to target nucleotides 2-10 ofthe mature miR-128-1 (SEQ NO:3), e.g., complementary to CACAGUGAA, e.g.,have the sequence TTCACTGTG (SEQ ID NO:9, which is the same asnucleotides 12-20 of SEQ NO:7).

In some embodiments of the methods described herein, the inhibitorynucleic acid comprises all or part of AAAGAGACCGGTTCACTGTGA (SEQ IDNO:7).

In some embodiments of the methods described herein, the inhibitorynucleic acid is an antisense oligonucleotide. In some embodiments, theantisense oligonucleotide comprises a sequence that is complementary toSEQ ID NO:3.

In some embodiments of the methods described herein, the inhibitorynucleic acid has one or more chemical modifications to the backbone orside chains as described herein. In some embodiments of the methodsdescribed herein, the inhibitory nucleic acid is an antagomir. In someembodiments of the methods described herein, the inhibitory nucleic acidhas at least one locked nucleotide, and/or has a phosphorothioatebackbone.

In some embodiments of the methods described herein, the inhibitorynucleic acid is an interfering RNA. In some embodiments, the interferingRNA is a small hairpin RNA (shRNA) or small interfering RNA (siRNA).

In some embodiments of the methods described herein, the inhibitorynucleic acid sequence inhibits post-transcriptional processing of SEQ IDNO:1 or 5.

In some embodiments of the methods described herein, the subject hasmetabolic syndrome or Type 2 diabetes.

In some embodiments of the methods described herein, the methods includeselecting a subject on the basis that they have metabolic syndrome orType 2 diabetes.

In some embodiments of the methods described herein, the methods includedetecting the presence of one or more alleles associated with increased,levels of miR-128 and/or predisposition to increased levels of serumlipids, and optionally selecting a subject on the basis of the presenceof an allele associated with increased levels of miR-128.

In some embodiments of the methods described herein, the methods includedetermining a level of triglycerides in the subject, and selecting thesubject if they have mildly elevated fasting levels (above 150 mg/dL(1.7 mmol/L)) or high fasting levels above 200 mg/dL (2.26 mmol/L)).

In some embodiments of the methods described herein, the methods includeselecting a subject who is in need of weight loss, a subject with a BMIof 25 or above.

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence capable of hybridizing with another nucleic acidsequence comprised of complementary nucleotide base pairs. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T) (or uracil (U) in the case of RNA), and guanine (G) forms abase pair with cytosine (C)) under suitable conditions of stringency.(See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For thepurposes of the present methods, the inhibitory nucleic acid need not becomplementary to the entire sequence, only enough of it to providespecific inhibition; for example in some embodiments the sequence is100% complementary to at least nucleotides (nts) 2-7 or 2-8 at the 5′end of the microRNA itself (e.g. the ‘seed sequence’), e.g., nts 2-7 or2-8 of SEQ ID NOs:2 or 3. Further details are provided below.

As used herein, an “antisense oligonucleotide” refers to a nucleic acidsequence that is complementary to a DNA or RNA sequence, such as that ofa microRNA.

“RNA” is a molecule comprising at least one or more ribonucleotideresidues. A “ribonucleotide” is a nucleotide with a hydroxyl group atthe 2′ position of a beta-D-ribofuranose moiety. The term RNA, as usedherein, includes double-stranded RNA, single-stranded RNA, isolated RNA,such as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Nucleotides of the RNA moleculescan also comprise non-standard nucleotides, such as non-naturallyoccurring nucleotides or chemically synthesized nucleotides ordeoxynucleotides.

A “microRNA” (miRNA) is a single-stranded RNA molecule of about 21-23nts in length. In general, miRNAs regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein. Each primary miRNA transcript is processed intoa short stem-loop structure (see, e.g., FIG. 3) before undergoingfurther processing into a functional miRNA. Mature miRNA molecules arepartially complementary to one or more messenger RNA (mRNA) molecules,and their main function is to down-regulate gene expression.

As used herein “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable—either directly or indirectly(i.e., upon conversion)—of inhibiting or down regulating gene expressionby mediating RNA interference. Interfering RNA includes but is notlimited to small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family.

A “small interfering RNA” or “siRNA” as used herein refers to any smallRNA molecule capable of inhibiting or down regulating gene expression bymediating RNA interference in a sequence specific manner. The small RNAcan be, for example, about 18 to 21 nucleotides long.

As used herein, an “antagomir” refers to a small synthetic RNA havingcomplementarity to a specific microRNA target, with either mispairing atthe cleavage site or one or more base modifications to inhibit cleavage.

As used herein, the phrase “post-transcriptional processing” refers tomRNA processing that occurs after transcription and is mediated, forexample, by the enzymes Dicer and/or Drosha.

By “an effective amount” is meant the amount of a required agent orcomposition comprising the agent to ameliorate the symptoms of a diseaserelative to an untreated patient. The effective amount of composition(s)used to practice the present invention for therapeutic treatment of adisease varies depending upon the manner of administration, the age,body weight, and general health of the subject. Ultimately, theattending physician or veterinarian will decide the appropriate amountand dosage regimen. Such amount is referred to as an “effective”amount.”

As used herein, “cholesterol homeostasis” refers to the regulation ofcholesterol uptake, cholesterol biosynthesis, cholesterol conversion tobile acids and excretion of bile acids as such processes occur in asubject having healthful levels of LDL, HDL and cholesterol in the blood(e.g., such healthful levels are also referred to herein as a “referencestandard”). Accordingly, a subject in need of cholesterol homeostasis isin need of improved regulation resulting in a return to healthful levelsof LDL, HDL and/or cholesterol in the blood.

A “subject” is a vertebrate, including any member of the class mammalia,including humans, domestic and farm animals, and zoo, sports or petanimals, such as mouse, rabbit, pig, sheep, goat, cattle and higherprimates. In preferred embodiments the subject is a human.

As used herein, a “vector” or “expression vector” is a nucleicacid-based delivery vehicle comprising regulatory sequences and a geneof interest, which can be used to transfer its contents into a cell.

Other definitions appear in context throughout this disclosure. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, sequences, database entries,and other references mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the sequence of human miR-128-1 precursor, also known asmiR-128A precursor (SEQ ID NO:1).

FIG. 2 shows the sequence of mature human miR-128 DNA (SEQ ID NO:2) andRNA (SEQ NO:3). The mature sequences are the same for miR-128-1 andmiR-128-2.

FIG. 3 shows the predicted hairpin structure of miR-128-1 precursor RNA(SEQ ID NO:4).

FIG. 4 shows the sequence of human miR-128-2 precursor, also known asmiR-128B precursor (SEQ NO:5).

FIG. 5 shows the predicted hairpin structure of miR-128-2 precursor RNA(SEQ ID NO:6).

FIG. 6 shows a schematic representation of the RAB3GAP1-R3HDM1-LCTgenomic locus harboring miR-128-1 on human chromosome 2. miR-128-1 islocated in intron 18 of R3HDM1. The depicted 1.23 Mb genomic regioncontains the SNPs associated with elevated LDL-C and TC in the analysesin Ma et al.

FIGS. 7A-B are bar graphs showing expression profiles of the miR-128-1host gene R3HDM1 (7A) and miR-128 (7B) in human tissues. Both genes areshown to be expressed fairly ubiquitously. To the best of the presentinventors' knowledge, the function of R3HDM1 has not yet beendetermined. Additionally, the expression profile of miR-128 reflects thecomposite expression of both miR-128-1 (located in the R3HDM1 gene) andmiR-128-2, whose genomic location is within the ARPP-21 host gene.miR-128-1 and miR-128-2 have identical mature sequences, which aremeasured here.

FIGS. 8A-B each show the immunoblotting results of three separateexperiments demonstrating that introduction of antisenseoligonucleotides complementary to human miR-128-1 into human HepG2 livercells causes increased expression of LDLR (8A), and introduction ofhuman miR-128-1 precursor oligonucleotides into human HepG2 liver cellscauses decreased expression of LDLR (8B).

FIGS. 9A-B are each immunoblots showing that introduction of antisenseoligonucleotides complementary to human miR-128-1 into human HepG2 livercells causes increased expression of ABGA1 (9A), and introduction ofhuman miR-128-1 precursor oligonucleotides into HepG2 cells causesdecreased expression of ABCA1 (9B). Thus, miR-128-1 is shown to controlthe expression of ABCA1.

FIGS. 10A-B are each immunoblots showing that introduction of antisenseoligonucleotides complementary to human miR-128-1 into human HepG2 livercells causes increased expression of SIRT1 (10A), and introduction ofhuman miR-128-1 precursor oligonucleotides into HepG2 cells results indecreased expression of SIRT1 (10B).

FIGS. 11A-C are immunoblots showing regulation of IRS1 and CYP39A1expression by miR-128-1. Introduction of antisense oligonucleotidescomplementary to human miR-128-1 into HepG2 cells causes increasedexpression of IRS1 (11A), and introduction of human miR-128-1 precursoroligonucleotides into HepG2 cells causes decreased expression of IRS1(11B) and decreased expression of CYP39A1 (11C).

FIGS. 12A-B are bar graphs showing miR-128-1 target validation atLDLR-3′UTR. Insertion of the LDLR-3′UTR sequence into a Luciferasereporter construct results in strongly decreased luciferase expressionin HEK293 cells, suggesting the presence of repressive regulatory motifswithin the LDLR-3′UTR (12A). Introduction of human miR-128-1 causesfurther repression of the Luciferase-LDLR 3′UTR, showing that LDLR isspecifically targeted by miR-128-1 through its 3′ UTR (12B).

FIGS. 13A-B are bar graphs showing that MiR-128-1 targets the ABCA1(13A) and SIRT1 (13B) 3′ UTRs for post-transcriptional regulation.Introduction of human miR-128-1 causes a repression of of theLuciferase-ABCA1 and SIRT1 3′UTRs, showing specific targeting bymiR-128-1 through their 3′UTR.

FIGS. 14A-B are bar graphs showing that miR-128-1-mediated regulation ofLDL receptor expression affects LDL uptake in human hepatoma cells.Fluorescently labeled (Dil) LDL uptake is strongly reduced upontreatment of human HepG2 hepatoma cells with miR-128-1 precursors (14A),whereas a modest but significant increase in Dil-LDL uptake is observedafter miR-128-1 antisense treatment (14B). All experiments were repeatedat least three times.

FIG. 15 is an immunoblot showing that miR-128-1 regulates ABCA1 andLDLR, which are involved in cholesterol/lipid and energy homeostasis, inthe human liver cell line Huh-7. miR-128-1 antisense treatment of Huh-7cells resulted in elevated expression of ABCA1 and LDLR. Beta-tubulinwas used as negative control.

FIG. 16 shows the results of Genome-wide analysis of miR-128-1biological effect on lipoprotein metabolism genes in HepG2 human livercells. As expected, excess miR-128-1 decreases the level of its targetgene LDLR, however, miR-128-1 also negatively affects the expression ofmajor protein components of HDL lipoprotein particles such as ApoA1,ApoC1-3 and ApoE, indicating the disruptive role of miR-128-1 in properlipid metabolism and trafficking. Interestingly, this indirect effect isextended to an induced expression of ApoB, a major component of VLDL andLDL lipoproteins (“bad cholesterol”). Unbiased hierarchical clusteringanalysis was applied to the DNA microarray data.

FIGS. 17A-B are line graphs showing that lentiviral-mediatedover-expression of miR-128-1 increases the LDL/HDL ratio in mice. FPLCanalysis of cholesterol-containing lipoprotein (HDL and LDL) profiles inpooled serum samples from 5-10 mice each in two separate experimentsindicate that ectopic miR-128-1 expression alters the distribution ofserum HDL and LDL particles while total plasma cholesterol levels werenot affected.

FIG. 18 shows the miR-128-1 precursor sequence (bold) including themiR-128-1 guide-strand sequence (underlined) flanked by 200 nt on eachside, SEQ ID NO:8.

DETAILED DESCRIPTION

The bioinformatics analyses and experimental evidence presented hereinthat miR-128-1 targets genes involved in cholesterol/lipid homeostasisand insulin signaling/energy homeostasis, suggest that theR3HDM1/miR-128-1 genomic locus may harbor a “thrifty” gene whoseelevated expression would allow increased fat storage/energyconservation in the face of starvation, thus providing a survivaladvantage in lean times. However, increased activity of such a thriftygene (or thrifty microRNA in this case) would also cause excess fatstorage when food/energy resources are plenty, and could potentiallycontribute to Metabolic Syndrome, type 2 diabetes, and cardiovasculardisease (CVD) in a subset of human populations harboring predisposingSNP alleles.

The evidence indicates that the miR-128-1 microRNA may target a numberof key regulators of cholesterol/lipid homeostasis and insulinsignaling/energy homeostasis see Table 1); validation evidence presentedherein demonstrate regulation of genes including the low-densitylipoprotein receptor (LDLR), the ATP-binding cassette A1 transporter(ABCA1) which is critical for high-density lipoprotein (HDL) synthesisand reverse cholesterol transport, the key regulator of lipid/energyhomeostasis SIRT1, the insulin signaling intermediate IRS1, and theCYP39A1 enzyme which converts cholesterol to bile for biliary excretion.

TABLE 1 Predicted targets of miR-128-1 involved in cholesterol/lipidhomeostasis and insulin signaling based on TargetScan Software Program.Shown in bold are genes verified as miR-128-1 targets in experiments.Cholesterol & Fatty acids Homeostasis Target genes LDL receptors andLDLR IRS1 Insulin Signaling associated proteins LDLRAP1 INSR PCSK9 INSM1CXCL16 IGF1 LRP6 IGFR1 STAB2 IGF2BP3 COLEC12 SIRT1 Fat/Lipid & EnergyCholesterol Transport ABCA1 FOXO1 Key Regulatory ABCG1 PPARgamma FactorsApoF PPARalpha ApoOL PRKAA2 ApoL6 PRKAG2 ApoL2 LEP ApoLD1 ADIPOQCholesterol/Lipid FDFT1 metabolism DHCR24 LIPA LPIN1 OSBPL10 OSBPL2OSBPL5 Cholesterol Cyp39A1 Catabolism Cyp7B1 Cyp8B1 Fatty acidmetabolism ACAA2 FAR1 FAR2 FADS1 ELOVL7 ELOVL6 ELOVL1 ELOVL2

Thus, described herein are methods using miR-128-1 antisense treatment,e.g., in subjects suffering from Metabolic Syndrome (e.g., high LDL, lowHDL, high triglycerides, obesity, nonalcoholic fatty liver disease,insulin resistance, and/or hypertension), type 2 diabetes, and/orcardiovascular disease (CVD). Such treatment is expected to result inone or more of the following:

a. lowering of circulating LDL (due to increased clearance by elevatedhepatic LDLR and CYP39A1);

b. increased HDL (due to elevated expression of ABCA1 in liver andperipheral tissues);

c. lowered triglycerides (due to increased hepatic expression of SIRT1and improved insulin signaling);

d. decreased obesity (due to increased adipose expression of SIRT1,Leptin and adiponectin);

e. decreased insulin resistance and type 2 diabetes (due to improvedinsulin signaling caused by increased expression of INSR, IRS1, andISL1);

f. ameliorated nonalcoholic fatty liver disease (due to increasedhepatic expression of SIRT1); and/or

g. decreased atherosclerosis/CVD (due to increased hepatic LDLR, ABCA1,SIRT1, and CYP39A1 expression, as well as increased ABCA1 expression inatherogenic macrophages/foam cells associated with increased cholesterolefflux and reverse cholesterol transport to the liver).

Because of the predicted and demonstrated impact of miR-128-1 on anumber of key regulators of cholesterol/lipid homeostasis and insulinsignaling/energy homeostasis, without wishing to be bound by theory, itis hypothesized that miR-128-1 may itself represent a central regulatorof human metabolism rivaling transcription factors such as SREBPs, LXRs,PPARs, and CREB in governing diverse metabolic circuits.

Bioinformatic Identification of a Potential Role for miR-128

The miR-128-1 microRNA is located in intron 18 of the R3HDM1 gene onhuman chromosome 2. Single nucleotide polymorphisms (SNPs) in a roughly1 Mb genomic region in and surrounding the R3HDM1/miR-128-1 locus havebeen associated with increased total cholesterol and LDL-cholesterol ingenome-wide association studies (GWAS), including in the FraminghamHeart Study and in a meta-analysis of 46 GWAS linking 95 genomic loci tovarious blood lipid parameters in >100,000 people. Ma et al., BMCMedical Genetics 11:55 (2010), identified multiple SNPs in a 1.23 Mbgenomic region as significantly associated with high LDL, and totalcholesterol in >6,000 participants in the Framingham Heart Study. Two ofthe SNPs (rs12465802 and rs4954280) are located in the miR-128-1 hostgene R3HDM1 (including in intron 18, within about 2 Kb of miR-128-1);see Table 1 and FIG. 2 of Ma et al, Teslovich et al., Nature 466:707-713(2010), performed GWAS of >100,000 individuals of European descent;among the 95 SNPs identified in their screen was a SNP (rs7570971) inthe RAB3GAP1-R3HDM1-LCT locus; the SNP was associated with elevatedtotal cholesterol (see Table 1 of Teslovich et al). Silander et al.,PLoS One 3:e3615 (2008), found an association between variants in thelactase (LCT) gene within the RAB3GAP1-R3HDM1-LCT genomic locus and bothtotal and LDL-cholesterol (e.g., rs4988235, rs6719488, rs619054, rs7412,rs4988235, and rs6719488, see Table 5 of Silander et al.). Haplotypeanalysis implied that the associated variants are in the LCT geneitself, and not necessarily related to the lactase persistence variantupstream of the gene. The C of the exonic variant rs2304371, which wasassociated with highest cholesterol values, is the ancestral allele,present in other mammals and located in a highly conserved region.

Many SNPs in this genomic locus have also been linked to naturalselection in a number of human populations irrespective of geographiclocation, Sabeti et al., Nature 449:913-8 (2007), identified theRAB3GAP1-R3HDM1-LCT genomic locus as positively selected during humanevolution. 24 SNPs were identified in this 2.4 Mb genomic locus based onthe fulfillment of two criteria: (1) selected alleles detectable by thetests are likely to be derived (newly arisen), because long-haplotypetests have little power to detect selection on standing (pre-existing)variation; the study was therefore focused on derived alleles, asidentified by comparison to primate outgroups; (2) selected alleles arelikely to be highly differentiated between populations, because recentselection is probably a local environmental adaptation; the study thuslooked for alleles common in only the population(s) under selection.Muiñios-Gimeno et al., Eur. J. Hum. Genet. 18; 218-26 (2010),investigated the possible association of 325 distinct human microRNAswith predisposition to disease. SNP coverage analysis revealed a lowerSNP density in miRNAs compared with the average of the genome, with only24 SNPs located in the 325 miRNAs studied. Further genotyping of 340unrelated Spanish individuals showed that more than half of the SNPsmiRNAs were either rare or monomorphic, in agreement with the reportedselective constraint on human miRNAs. A comparison of the minor allelefrequencies between Spanish and HapMap population samples confirmed theapplicability of this SNP panel to the study of complex disorders amongthe Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 inthe CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showinggeographical allelic frequency variation among the four HapMappopulations, probably because of differences in natural selection. TheBovine HapMap Consortium, Science 324:528-32(2009), revealed a link ofSNPs in the R3HDM1 genomic locus (e.g., rs29021800) to intramuscular fat(marbling) and “feed efficiency”, i.e. cattle with this genotype havelikely been selected for the trait to thrive on less feed; they consumeless energy per amount of food given. This is a hallmark of a proposed“thrifty gene.”

However, the fact that in addition to several genes this locus harbors amicroRNA with targets that participate in regulation ofcholesterol/lipid/energy homeostasis was not previously noted. MicroRNAs(miRNAs) are a class of small (e.g., 18-24 nucleotides) non-coding RNAsthat exist in a variety of organisms, including mammals, and areconserved in evolution, miRNAs are processed from hairpin precursors ofabout 70 nucleotides which are derived from primary transcripts throughsequential cleavage by the RNAse III enzymes drosha and dicer. ManymicroRNAs can be encoded in intergenic regions, hosted within introns ofpre-mRNAs or within ncRNA genes. Many miRNAs also tend to be clusteredand transcribed as polycistrons and often have similar spatial temporalexpression patterns. MiRNAs have been found to have roles in a varietyof biological processes including developmental timing, differentiation,apoptosis, cell proliferation, organ development, and metabolism.

Methods of Treatment

The methods described herein include the inhibition miR-128 in a subjectwho has cholesterol/lipid abnormalities (e.g., elevated circulating LDL,low HDL, elevated triglycerides), and/or is insulin resistant, and/orhas the metabolic syndrome, and/or is suffering from type 2 diabetes,and/or cardiovascular disease. This can be achieved, for example, byadministering an inhibitory nucleic acid, e.g., an antisenseoligonucleotide that is complementary to miR-128, including but notlimited to an antisense oligonucleotide comprising all or part ofAAAGAGACCGGTTCACTGTGA (SEQ ID NO:7); in some embodiments, as describedin farther detail below, the oligo includes different modifications,e.g., in the sugar backbone, to make it more cell permeable and nucleaseresistant on one hand, and physiologically non-toxic at lowconcentrations on the other. Other inhibitory nucleic acids for use inpracticing the methods described herein and that are complementary tomiR-128 can be those which inhibit post-transcriptional processing ofmiR-128, such as an interfering RNA, including but not limited to anshRNA or siRNA, or an antagomir.

Inhibitory Nucleic Acids

Inhibitory nucleic acids useful in the present methods and compositionsinclude antisense oligonucleotides, ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, modifiedbases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids(PNAs), and other oligomeric compounds or oligonucleotide mimetics whichhybridize to at least a portion of the target nucleic acid (i.e.,miR-128, e.g., all or part of any of SEQ ID NOs:1-6) and modulate itsfunction. In some embodiments, the inhibitory nucleic acids includeantisense RNA, antisense DNA, chimeric antisense oligonucleotides,antisense oligonucleotides comprising modified linkages, interferenceRNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA);small RNA-induced gene activation (RNAa); small activating RNAs(saRNAs), or combinations thereof. See, e.g., WO 2010040112.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 13 to50, or 13 to 30 nucleotides in length. One having ordinary skill in theart will appreciate that this embodies oligonucleotides having antisenseportions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any rangetherewithin. In some embodiments, the oligonucleotides are 15nucleotides in length. In some embodiments, the antisense oroligonucleotide compounds of the invention are 12 or 13 to 30nucleotides in length. One having ordinary skill in the art willappreciate that this embodies inhibitory nucleic acids having antisenseportions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 nucleotides in length, or any range therewithin.

In some embodiments, the inhibitory nucleic acids are designed to targeta specific region of miR-128. For example, a specific functional regioncan be targeted, e.g., a region comprising a seed sequence or a regioncomplementary to the target nucleic acid on which the miR-128 acts. Forexample, the inhibitory nucleic acid can be designed to targetnucleotides 2-10 of the mature miR-128-1, e.g., complementary toCACAGUGAA, e.g., have the sequence TTCACTGTG (SEQ ID NO:9, which is thesame as nucleotides 12-20 of SEQ ID NO:7). Alternatively or in addition,highly conserved regions can be targeted, e.g., regions identified byaligning sequences from disparate species such as primate (e.g., human)and rodent (e.g., mouse) and looking for regions with high degrees ofidentity. Percent identity can be determined routinely using basic localalignment search tools (BLAST programs) (Altschul et al., Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656),e.g., using the default parameters.

In some embodiments, the inhibitory nucleic acids are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimericinhibitory nucleic acids of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acid comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Inother preferred embodiments, RNA modifications include 2′-fluoro,2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues or an inverted base at the 3′ end of the RNA. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than; 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide; thesemodified oligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholinobackbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506);peptide nucleic acid (PNA) backbone (wherein the phosphodiester backboneof the oligonucleotide is replaced with a polyamide backbone, thenucleotides being bound directly or indirectly to the aza nitrogen atomsof the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., Chem. Soc., 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sultanateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-0-CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)](Martin et al, HeIv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2′-methoxy (2′-0-CH₃), 2′-propoxy(2′-OCH₂CH₂CH₃) and 2′-fluoro (2T-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Inhibitory nucleic acids can also include, additionally, oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me—C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases,e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolyalkl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, 1980, pp 75-77; Gebeyehu, C., et al. Nucl. Acids Res.1987, 15:4513). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me—C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2<0>C. (Sanghvi, Y. S., inCrooke, S. T. and Lebleu, B., eds., Antisense Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferredbase substitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are hounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science, 1991, 254, 1497-1500.

Inhibitory nucleic acids can also include one or more nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases comprise thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Modified nucleobases compriseother synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications',pages 289-302, Crooke, S. T. and Leblett, B, ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications. Modifiednucleobases are described in U.S. Pat. No. 3,687,808, as well as U.S.Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acids are chemically linkedto one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Suchmoieties comprise but are not limited to, lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al, Ann. N. Y. Acad., Sci., 1992, 660, 306-309; Manoharan et al.,Bioorg, Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBSLett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J.Pharmacol. Exp. Ther., 1996, 277, 923-937). See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluorescents, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, dodecandiol or undecyl residues,a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See,e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941.

The inhibitory nucleic acids useful in the present methods aresufficiently complementary to all or part of miR-128, i.e., hybridizesufficiently well and with sufficient specificity, to give the desiredeffect. “Complementary” refers to the capacity for pairing, throughhydrogen bonding, between two sequences comprising naturally ornon-naturally occurring bases or analogs thereof. For example, if a baseat one position of an inhibitory nucleic acid is capable of hydrogenbonding with a base at the corresponding position of a miR-128 sequence,then the bases are considered to be complementary to each other at thatposition. 100% complementarity is not required.

In the context of this invention, hybridization means hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. Complementary, as used herein,refers to the capacity for precise pairing between two nucleotides. Theinhibitory nucleic acids and the miR-128 are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleotides that can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarily or precise pairingsuch that stable and specific binding occurs between the inhibitorynucleic acid and the miR-128 target sequence. For example, if a base atone position of an inhibitory nucleic acid is capable of hydrogenbonding with a base at the corresponding position of a miR-128 molecule,then the bases are considered to be complementary to each other at thatposition.

Although in some embodiments, 100% complementarity is desirable, it isunderstood in the art that a complementary nucleic acid sequence neednot be 100% complementary to that of its target nucleic acid to bespecifically hybridisable. A complementary nucleic acid sequence forpurposes of the present methods is specifically hybridisable whenbinding of the sequence to the target miR-128 molecule interferes withthe normal function of the target miR-128 to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the sequence to non-target miR-128 sequencesunder conditions in which specific binding is desired, e.g., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed under suitable conditions of stringency. Forexample, stringent salt concentration will ordinarily be less than about750 mM NaCl and 75 trisodium citrate, preferably less than about 500 mMNaCl and 50 mM trisodium citrate, and more preferably less than about250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridizationcan be obtained in the absence of organic solvent, e.g., formamide,while high stringency hybridization can be obtained in the presence ofat least about 35% formamide, and more preferably at least about 50%formamide. Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. Varying additionalparameters, such as hybridization time, the concentration of detergent,e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion ofcarrier DNA, are well known to those skilled in the art. Various levelsof stringency are accomplished by combining these various conditions asneeded. In a preferred embodiment, hybridization will occur at 30° C. in750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferredembodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mMtrisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmonsperm DNA (ssDNA). In a most preferred embodiment, hybridization willoccur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50%formamide, and 200 μg/ml ssDNA. Useful variations on these conditionswill be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42″ C. in 15 nM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods describedherein have at least 80% sequence complementarity to a target regionwithin the target nucleic acid, e.g., 90%, 95%, or 100% sequencecomplementarity to the target region within miR-128 (e.g., a targetregion comprising the seed sequence). For example, an antisense compoundin which 18 of 20 nucleobases of the antisense oligonucleotide arecomplementary, and would therefore specifically hybridize, to a targetregion would represent 90 percent complementarily. Percentcomplementarity of an inhibitory nucleic acid with a region of a targetnucleic acid can be determined routinely using basic local alignmentsearch tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990,215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).Antisense and other compounds of the invention that hybridize to amiR-128 target sequence are identified through routine experimentation.In general the inhibitory nucleic acids must retain specificity fortheir target, i.e., must not directly bind to, or directly significantlyaffect expression levels of, transcripts other than the intended target.

For further disclosure regarding inhibitory nucleic acids, please seeUS2010/0317718 (antisense oligos); US2010/0249052 (double-strandedribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs);US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); andWO2010/129746 and WO2010/040112 (inhibitory nucleic acids).

Antisense

In some embodiments, the inhibitory nucleic acids are antisenseoligonucleotides. Antisense oligonucleotides are typically designed toblock expression of a DNA or RNA target by binding to the target andhalting expression at the level of transcription, translation, orsplicing. Antisense oligonucleotides of the present invention arecomplementary nucleic acid sequences designed to hybridize understringent conditions to a miR-128 target sequence. Thus,oligonucleotides are chosen that are sufficiently complementary to thetarget, i.e., that hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

Modified Bases/Locked Nucleic Acids (LNAs)

In some embodiments, the inhibitory nucleic acids used in the methodsdescribed herein comprise one or more modified bonds or bases. Modifiedbases include phosphorothioate, methylphosphonate, peptide nucleicacids, or locked nucleic acid (LNA) molecules. Preferably, the modifiednucleotides are locked nucleic acid molecules, including [alpha]-L-LNAs.LNAs comprise ribonucleic acid analogues wherein the ribose ring is“locked” by a methylene bridge between the 2′-oxgygen and the4′-carbon—i.e., oligonucleotides containing at least one LNA monomer,that is, one 2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide. LNA basesform standard Watson-Crick base pairs but the locked configurationincreases the rate and stability of the basepairing reaction (Jepsen etal., Oligonucleotides, 14, 130-146 (2004)). LNAs also have increasedaffinity to base pair with RNA as compared to DNA. These propertiesrender LNAs especially useful as probes for fluorescence in situhybridization (FISH) and comparative genomic hybridization, as knockdowntools for miRNAs, and as antisense oligonucleotides to target mRNAs orother RNAs.

The LNA molecules can include molecules comprising 10-30, 12-24, e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides in each strand, wherein one of the strands issubstantially identical, e.g., at least 80% (or more, e.g., 85%, 90%,95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a miR-128 target sequence. The LNA molecules can bechemically synthesized using methods known in the art.

The LNA molecules can be designed using any method known in the art; anumber of algorithms are known, and are commercially available (e.g., onthe internet, for example at exiqon.com). See, e.g., You et al., Nuc.Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43:5388-405(2004); and Levin et al., Nuc. Acids. Res. 34:e142 (2006). For example,“gene walk” methods, similar to those used to design antisense oligos,can be used to optimize the inhibitory activity of the LNA (or any otherinhibitory nucleic acid described herein); for example, a series ofoligonucleotides of 10-30 nucleotides spanning the length of a targetmiR-128 sequence can be prepared, followed by testing for activity.Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left betweenthe LNAs to reduce the number of oligonucleotides synthesized andtested. GC content is preferably between about 30-60%. Generalguidelines for designing LNAs are known in the art; for example, LNAsequences will bind very tightly to other LNA sequences, so it ispreferable to avoid significant complementarity within an LNA.Contiguous runs of three or more Gs or Cs, or more than four LNAresidues, should be avoided where possible (for example, it may not bepossible with very short (e.g., about 9-10 nt) oligonucleotides). Insome embodiments, the LNAs are xylo-LNAs.

For additional information regarding LNAs see U.S. Pat. Nos. 6,268,490;6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,060,809;7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos. 20100267018;20100261175; and 20100035968; Koshkin et al. Tetrahedron 54, 3607-3630(1998); Obika et al. Tetrahedron Lett. 39, 5401-5404 (1998); Jepsen etal., Oligonucleotides 14:130-A46 (2004); Kauppinen et al., Drug Disc.Today 2(3):287-290 (2005); and Ponting et al., Cell 136(4):629-641(2009), and references cited therein.

In some embodiments of the methods described herein, the inhibitorynucleic acid is or comprises TTCACTGTG (SEQ ID NO:9), wherein all of thenucleic acids are locked and the backbone is a phosphorothioatebackbone.

Antagomirs

In some embodiments, the antisense is an antagomir. Antagomirs arechemically modified antisense oligonucleotides that target a miR-128target sequence. For example, an antagomir for use in the methodsdescribed herein can include a nucleotide sequence sufficientlycomplementary to hybridize to a miR-128 target sequence of about 12 to25 nucleotides, preferably about 15 to 23 nucleotides.

In general, antagomirs include a cholesterol moiety, e.g., at the3′-end. In some embodiments, antagomirs have various modifications forRNase protection and pharmacologic properties such as enhanced tissueand cellular uptake. For example, In addition to the modificationsdiscussed above for antisense oligos, antagomir can have one or more ofcomplete or partial 2′-O-methylation of sugar and/or a phosphorothioatebackbone. Phosphorothioate modifications provide protection againstRNase activity and their lipophilicity contributes to enhanced tissueuptake. In some embodiments, the antagomir can include sixphosphorothioate backbone modifications; two phosphorothioates arelocated at the 5′-end and four at the 3′-end. See, e.g., Krutzfeldt etal., Nature 438, 685-689 (2005); Czech, N Engl J Med 2006; 354:1194-1195(2006); Robertson et al., Silence. 1:10 (2010); Marquez and McCaffrey,Hum Gene Ther. 19(1):27-38 (2008); van Rooij et al., Circ Res.103(9):919-928 (2008); and Liu et al., Int. J. Mol. Sci. 9:978-999(2008). Antagomirs useful in the present methods can also be modifiedwith respect to their length or otherwise the number of nucleotidesmaking up the antagomir. The antagomirs must retain specificity fortheir target, i.e., must not directly bind to, or directly significantlyaffect expression levels of transcripts other than the intended target.

In some embodiments, the inhibitory nucleic acid is locked and includesa cholesterol moiety (e.g., a locked antagomir).

siRNA/shRNA

In some embodiments, the nucleic acid sequence that is complementary tomiR-128 can be an interfering RNA, including but not limited to a smallinterfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”). Methods forconstructing interfering RNAs are well known in the art. For example,the interfering RNA can be assembled from two separate oligonucleotides,where one strand is the sense strand and the other is the antisensestrand, wherein the antisense and sense strands are self-complementary(i.e., each strand comprises nucleotide sequence that is complementaryto nucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure); theantisense strand comprises nucleotide sequence that is complementary toa nucleotide sequence in a target nucleic acid molecule or a portionthereof (i.e., an undesired gene) and the sense strand comprisesnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. Alternatively, interfering RNA is assembled from asingle oligonucleotide, where the self-complementary sense and antisenseregions are linked by means of nucleic acid based or non-nucleicacid-based linker(s). The interfering RNA can be a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises a nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The interfering can be a circular single-strandedpolynucleotide having two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure, and is referred to herein as an “shRNA.”The loop region is generally between about 2 and about 10 nucleotides inlength. In some embodiments, the loop region is from about 6 to about 9nucleotides in length. In some embodiments, the sense region and theantisense region are between about 15 and about 20 nucleotides inlength. Following post-transcriptional processing, the small hairpin RNAis converted into a siRNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. The siRNA is thencapable of inhibiting the expression of a gene with which it shareshomology. For details, see Brummelkamp et al., Science 296:550-553,(2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishiand Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes &Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002);Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. ProcNatl Acad Sci USA 99:6047-6052, (2002).

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target nucleic acid are preferred for inhibition.However, 100% sequence identity between the siRNA and the target gene isnot required to practice the present invention. Thus the invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Alternatively,siRNA sequences with nucleotide analog substitutions or insertions canbe effective for inhibition. In general the siRNAs must retainspecificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target,

Ribozymes

Trans-cleaving enzymatic nucleic acid molecules can also be used; theyhave shown promise as therapeutic agents for human disease (Usman &McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen andMarr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acidmolecules can be designed to cleave miR-128 within the background ofcellular RNA. Such a cleavage event renders the miR-128 non-functional.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution strategies(Orgel, 1979. Proc. R. Soc. London, B 205, 435) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker etal, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261:1411-1418;Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183;Breaker, 1996, Curr. Op. Biotech., 1, 442)

Making and Using Inhibitory Nucleic Acids

The nucleic acid sequences used to practice the methods describedherein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybridsthereof, can be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant nucleic acid sequences can be individually isolated orcloned and tested for a desired activity. Any recombinant expressionsystem can be used, including e.g. in vitro, bacterial, fungal,mammalian, yeast, insect or plant cell expression systems.

Nucleic acid sequences of the invention can be inserted into deliveryvectors and expressed from transcription units within the vectors. Therecombinant vectors can be DNA plasmids or viral vectors. Generation ofthe vector construct can be accomplished using any suitable geneticengineering techniques well known in the art, including, withoutlimitation, the standard techniques of PCR, oligonucleotide synthesis,restriction endonuclease digestion, ligation, transformation, plasmidpurification, and DNA sequencing, for example as described in Sambrooket al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al.(Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J.Cann, Ed., Oxford University Press, (2000)). As will be apparent to oneof ordinary skill in the art, a variety of suitable vectors areavailable for transferring nucleic acids of the invention into cells.The selection of an appropriate vector to deliver nucleic acids andoptimization of the conditions for insertion of the selected expressionvector into the cell, are within the scope of one of ordinary skill inthe art without the need for undue experimentation. Viral vectorscomprise a nucleotide sequence having sequences for the production ofrecombinant virus in a packaging cell. Viral vectors expressing nucleicacids of the invention can be constructed based on viral backbonesincluding, but not limited to, a retrovirus, lentivirus, adenovirus,adeno-associated virus, pox virus or alphavirus. The recombinant vectorscapable of expressing the nucleic acids of the invention can bedelivered as described herein, and persist in target cells (e.g., stabletransformants).

Nucleic acid sequences used to practice this invention can besynthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Nucleic acid sequences of the invention can be stabilized againstnucleolytic degradation such as by the incorporation of a modification,e.g., a nucleotide modification. For example, nucleic acid sequences ofthe invention includes a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA). As another example, the nucleic acidsequence can include at least one 2-O-methyl-modified nucleotide, and insome embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom (see,e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin etal., J. Am. Chem. Soc., 120(50):13252-13253 (1998)). For additionalmodifications see US 20100004320, US 20090298916, and US 20090143326.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual 3d ed. (2001); CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,Inc., New York 2010); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); Laboratory Techniques In BiochemistryMolecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Pharmaceutical Compositions

The methods described herein can include the administration ofpharmaceutical compositions and formulations comprising inhibitorynucleic acid sequences designed to target miR-128.

In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, topically, orally or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

The inhibitory nucleic acids can be administered alone or as a componentof a pharmaceutical formulation (composition). The compounds may beformulated for administration, in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

Formulations of the compositions of the invention include those suitablefor intradermal, inhalation, oral/nasal, topical, parenteral, rectal,and/or intravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,nucleic acid sequences of this invention) which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration,e.g., intradermal or inhalation. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect, e.g., an antigen specific T cell or humoralresponse.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents and preserving agents. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums arabic and tragacanth; and proteins, e.g., gelatin and collagen.Disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acidsequences of the invention) in admixture with excipients suitable forthe manufacture of aqueous suspensions, e.g., for aqueous intradermalinjections. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

In some embodiments, oil-based pharmaceuticals are used foradministration of nucleic acid sequences of the invention. Oil-basedsuspensions can be formulated by suspending an active agent in avegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

Pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described above, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean so lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent. In alternativeembodiments, these injectable oil-in-water emulsions of the inventioncomprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitanmonooleate and/or an ethoxylated sorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal,intraocular and intravaginal routes including suppositories,insufflation, powders and aerosol formulations (for examples of steroidinhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193;Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In some embodiments, the pharmaceutical compounds can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

In some embodiments, the pharmaceutical compounds can also be deliveredas microspheres for slow release in the body. For example, microspherescan be administered via intradermal injection of drug which slowlyrelease subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed.7:623-645; as biodegradable and injectable gel formulations, see, e.g.,Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oraladministration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In some embodiments, the pharmaceutical compounds can be parenterallyadministered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations canbe lyophilized. Stable lyophilized formulations comprising an inhibitorynucleic acid can be made by lyophilizing a solution comprising apharmaceutical of the invention and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. A process forpreparing a stable lyophilized formulation can include lyophilizing asolution about 2.5 mg/mL protein, about 15 mg/mL, sucrose, about 19mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 butless than 6.5. See, e.g., U.S. 20040028670.

The compositions and formulations can be delivered by the use ofliposomes. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306;Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J.Hosp. Pharm. 46:1576-1587. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in abilayer or bilayers. Liposomes are unilamellar or multilamellar vesiclesthat have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes that are believed to interactwith negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically stabilized” liposomes, i.e.,liposomes comprising one or more specialized lipids. When incorporatedinto liposomes, these specialized lipids result in liposomes withenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860.

The formulations of the invention can be administered for prophylacticand/or a therapeutic treatments. In some embodiments, for therapeuticapplications, compositions are administered to a subject who is need ofreduced triglyceride levels, or who is at risk of or has a disorderdescribed herein, in an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of the disorder or itscomplications; this can be called a therapeutically effective amount.For example, in some embodiments, pharmaceutical compositions of theinvention are administered in an amount sufficient to decrease serumlevels of triglycerides in the subject.

The amount of pharmaceutical composition adequate to accomplish this isa therapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;Remington: The Science and Practice of Pharmacy, 21st ed., 2005). Thestate of the art allows the clinician to determine the dosage regimenfor each individual patient, active agent and disease or conditiontreated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, the degree and amount of therapeutic effectgenerated after each administration (e.g., effect on tumor size orgrowth), and the like. The formulations should provide a sufficientquantity of active agent to effectively treat, prevent or ameliorateconditions, diseases or symptoms.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in topical or oral administration or administering by powders,spray or inhalation. Actual methods for preparing parenterally ornon-parenterally administrable formulations will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington: The Science and Practice of Pharmacy, 21sted., 2005.

Various studies have reported successful mammalian dosing usingcomplementary nucleic acid sequences. For example, Esau C., et al.,(2006) Cell Metabolism, 3(2):87-98 reported dosing of normal mice withintraperitoneal doses of miR-122 antisense oligonucleotide ranging from12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy andnormal at the end of treatment, with no loss of body weight or reducedfood intake. Plasma transaminase levels were in the normal range (AST ¾45, ALT ¾ 35) for all doses with the exception of the 75 mg/kg dose ofmiR-122 ASO, which showed a very mild increase in ALT and AST levels.They concluded that 50 mg/kg was an effective, non-toxic dose. Anotherstudy by Krützfeldt J., et al., (2005) Nature 438, 685-689, injectedantagomirs to silence miR-122 in mice using a total dose of 80, 160 or240 mg per kg body weight. The highest dose resulted in a complete lossof miR-122 signal. In yet another study, locked nucleic acids (“LNAs”)were successfully applied in primates to silence miR-122. Elmen J., etal., (2008) Nature 452, 896-899, report that efficient silencing ofmiR-122 was achieved in primates by three doses of 10 mg kg-1LNA-anti-miR, leading to a long-lasting and reversible decrease in totalplasma cholesterol without any evidence for LNA-associated toxicities orhistopathological changes in the study animals.

In some embodiments, the methods described herein can includeco-administration with other drugs or pharmaceuticals, e.g.,compositions for providing cholesterol homeostasis. For example, theinhibitory nucleic acids can be co-administered with drugs for treatingor reducing risk of a disorder described herein.

Methods of Treatment

Also described herein are methods that can also be used to treatsubjects who have elevated triglycerides; are obese (BMI of 30 orhigher), pre-diabetic or diabetic; or who have the metabolic syndrome;thus, the methods can include detecting the presence of one of theseconditions, or diagnosing the subject with one of these conditions. Insome embodiments, the methods include detecting the presence of agenetic variant affecting miR-128 levels, and optionally selectingsubjects on the basis of the presence of such a variant.

Genetic Variants Affecting miR-128 Levels

In addition, the methods can also include identifying subjects fortreatment using a method described by detecting one or both of (i) thepresence of a genetic variation that predisposes a subject to elevatedmiR-128 levels, or (ii) the presence of elevated miR-128 levels, e.g.,in a sample comprising liver and/or white adipose cells or tissue, e.g.,from a biopsy.

In some embodiments, detecting the presence of a genetic variation thatpredisposes a subject to elevated miR-128 levels includes detecting theidentity of an allele of one, two or more of rs6730157, rs12465802,rs4954280, rs2322660, rs309180, rs632632, rs7570971, rs4988235, orrs6719488, wherein the presence of an allele that is associated withelevated levels of miR-128, or with increased risk of elevated lipidlevels (e.g., as described in Ma et al., BMC Medical Genetics 11:55(2010); Teslovich et al., Nature 466:707-713 (2010); and Silander etal., PLoS One 3:e3615 (2008)), indicates that the subject is predisposedto have elevated levels of miR-128, and thus would benefit from thetreatment methods described herein. In some embodiments, the methodsinclude selecting a subject for treatment with a method described hereinon the basis of the presence of an allele associated with elevatedlevels miR-128 or associated with increased risk of elevated lipidlevels.

Elevated Cholesterol and/or Triglycerides

Abnormal cholesterol and triglyceride levels are associated with risk ofdisease, including cardiovascular disease; in some embodiments, thesubjects treated using the methods described herein have, or are at riskof developing, abnormal cholesterol and/or triglyceride levels. In someembodiments, the subjects have elevated LDL-cholesterol, elevated totalcholesterol, and/or elevated triglyceride levels.

High Density Lipoprotein Low Density Lipoprotein (LDL) and Very LowDensity Lipoprotein (VLDL) are the three major kinds of cholesterol thatare monitored. Total cholesterol and cholesterol/HDL ratio can also bemonitored.

The following tables provide information regarding levels of cholesterolthat are considered to be optimal (i.e., associated with a low or normalrisk of cardiovascular disease) or abnormal (i.e., associated with ahigher risk of cardiovascular disease). The numbers are in milligramsper deciliter (mg/dL).

Elevated levels of LDL are associated with increased risk ofcardiovascular disease.

LDL Cholesterol LDL-Cholesterol Category Less than 100 Optimal 100-129Near optimal/above optimal 130-159 Borderline high 160-189 High 190 andabove Very high

In the presence of cardiovascular disease, some experts consider optimalLDL levels to be less than 70. For people with diabetes or othermultiple risk factors for heart disease, optimal levels of LDL are lessthan 100.

Increased levels of HDL cholesterol are associated with decreased riskof cardiovascular disease.

HDL Cholesterol HDL-Cholesterol Category 60 and above High; Optimal;lower risk of cardiovascular disease Less than 40 in men and less Low;increased risk factor of than 50 in women cardiovascular disease

Total blood cholesterol is a measure of LDL cholesterol, HDLcholesterol, and other lipid components combined. Total cholesterollevels below 200 are desirable.

Total Cholesterol Category Less than 200 Desirable 200-239 BorderlineHigh 240 and above High

Triglyceride (triacylglycerol, TAG or triacylglyceride) is an esterderived from glycerol and three fatty acids, and is the main constituentof vegetable oil and animal fats (Nelson, D. L.; Cox, M. M. Lehninger,Principles of Biochemistry, 3rd Ed. Worth Publishing: New York, 2000).

The American Heart Association has set guidelines for triglyceridelevels (after fasting for 8-12 hours), as follows:

Level (mg/dL) Level (mmoL/L) Interpretation <150 <1.69 Normal range, lowrisk 150-199 1.70-2.25 Borderline high 200-499 2.26-5.65 High >500 >5.65Very high: high risk

Fasting triglyceride levels can be determined using any means known inthe art, e.g., enzymatically using a glycerol kinase reaction-basedcolorimetric assay. Cholesterol levels can also be determined using anymeans known in the art, e.g., using immunoassay, electrophoresis, NMR,and/or precipitation-based methods.

Diabetic and Pre-Diabetic Subjects

In some embodiments, the subjects treated by the methods describedherein have diabetes, i.e., are diabetic. A person who is diabetic hasone or more of a Fasting Plasma Glucose Test result of 126 mg/dL ormore; a 2-Hour Plasma Glucose Result in an Oral Glucose Tolerance Testof 200 mg/dL or more; and blood glucose level of 200 mg/dL or above. Insome embodiments, the subjects treated by the methods described hereinare being treated for diabetes, e.g., have been prescribed or are takinginsulin, meglitinides, biguanides, thiazolidinediones, oralpha-glucosidase inhibitors.

In some embodiments the subjects are pre-diabetic, e.g., they haveimpaired glucose tolerance or impaired fasting glucose, e.g., asdetermined by standard clinical methods such as the intravenous glucosetolerance test (IVGTT) or oral glucose tolerance test (OGTT), e.g., avalue of 7.8-11.0 mmol/L two hours after a 75 g glucose drink forimpaired glucose tolerance, or a fasting glucose level (e.g., beforebreakfast) of 6.1-6.9 mmol/L.

The pathogenesis of type 2 diabetes is believed to generally involve twocore defects: insulin resistance and β-cell failure (Martin et al.,Lancet 340:925-929 (1992); Weyer et al., J. Clin. Invest. 104:787-794(1999); DeFronzo et al., Diabetes Care. 15:318-368 (1992)). Importantadvances towards the understanding of the development of peripheralinsulin resistance have been made in both animal models and humans(Bruning et al., Cell 88:561-572 (1997); Lauro et al., Nat. Genet.20:294-298 (1998); Nandi et al., Physiol. Rev. 84:623-647 (2004);Sreekumar et al., Diabetes 51:1913-1920 (2002); McCarthy and Froguel,Am. J. Physiol. Endocrinol. Metab. 283:E217-E225 (2002); Mauvais-Jarvisand Kahn, Diabetes, Metab. 26:433-448 (2000); Petersen et al., N. Engl.J. Med. 350:664-671 (2004)). Thus, those subjects who have or are atrisk for insulin resistance or impaired glucose tolerance are readilyidentifiable, and the treatment goals are well defined.

In some embodiments, the methods described herein include selectingsubjects who have diabetes or pre-diabetes. In some embodiments, thefollowing table is used to identify and/or select subjects who arediabetic or have pre-diabetes, i.e., impaired glucose tolerance and/orimpaired fasting glucose.

Fasting Blood Glucose From 70 to 99 mg/dL (3.9 to 5.5 mmol/L) Normalfasting glucose From 100 to 125 mg/dL (5.6 to 6.9 mmol/L) Impairedfasting glucose (pre-diabetes) 126 mg/dL (7.0 mmol/L) and above onDiabetes more than one testing occasion Oral Glucose Tolerance Test(OGTT) [except pregnancy] (2 hours after a 75-gram glucose drink) Lessthan 140 mg/dL (7.8 mmol/L) Normal glucose tolerance From 140 to 200mg/dL Impaired glucose tolerance (7.8 to 11.1 mmol/L) (pre-diabetes)Over 200 mg/dL (11.1 mmol/L) on more Diabetes than one testing occasion

Body Mass Index (BMI)

Obesity increases a subject's risk of developing T2D. BMI is determinedby weight relative to height, and equals a person's weight in kilogramsdivided by height in meters squared (BMI=kg/m²). Acceptedinterpretations are given in the following table:

Category BMI Underweight ≦18.5 Normal weight 18.5-24.9 Overweight  25-29.9 Obese ≧30

Thus, the methods described herein can include determining a subject'sheight, determining a subject's weight, and calculating BMI from thevalues determined thereby. Alternatively, the methods described hereincan include reviewing a subject's medical history to determine theirBMI.

In some embodiments, the methods described herein include selectingsubjects who have a BMI of 30 or above (i.e., obese subjects).

Metabolic Syndrome

In some embodiments, the methods include determining whether a subjecthas the metabolic syndrome, and selecting the subject if they do havethe metabolic syndrome, then administering an inhibitory nucleic acid asdescribed herein. Determining whether a subject has the metabolicsyndrome can include reviewing their medical history, or ordering orperforming such tests as are necessary to establish a diagnosis.

The metabolic syndrome, initially termed Syndrome X (Reaven, Diabetes,37(12):1595-1607 (1988)), refers to a clustering of obesity,dyslipidemia, hypertension, and insulin resistance. All components ofthe metabolic syndrome are traditional risk factors for vasculardisease. As used herein, the metabolic syndrome is defined by thepresence of at least 3 of the following: abdominal obesity (excessivefat tissue in and around the abdomen, as measured by waistcircumference: e.g., greater than 40 inches for men, and greater than 35inches for women), fasting blood triglycerides (e.g., greater than orequal to 150 mg/dL), low HDL (e.g., less than 40 mg/dL for men, and lessthan 50 mg/DL for women), elevated LDL (e.g., above 130 mg/dL), highblood pressure (e.g., greater than or equal to 130/85 mmHg) and/orelevated fasting glucose (e.g., greater than or equal to 110 mg/dL). Insome embodiments, levels of these criteria may be higher or lower,depending on the subject; for example, in subjects of Asian ancestry;see, e.g., Meigs, Curr. Op. Endocrin. Diabetes, 13(2):103-110 (2006). Adetermination of the presence of metabolic syndrome can be made, byreviewing the subject's medical history, or by reviewing test results.

Based on data from the Third National Health and Nutrition ExaminationSurvey (NHANES III) approximately 24% of the adults in the United Statesqualify as having the metabolic syndrome (Ford et al., JAMA.287(3):356-359 (2002)). Insulin resistance is now felt to be central inthe pathogenesis of these related disorders.

Nonalcoholic fatty liver disease (NAFLD) and its most severe form,nonalcoholic steatohepatitis (NASH), are associated with high fat diet,high triglyceride levels, obesity, the metabolic syndrome and type 2diabetes, and pose an increased risk of cardiovascular disease. NAFLD isan accumulation of fat in the liver that is not a result of excessiveconsumption of alcohol. 15% to 25% of cases of NAFLD progress and areassociated with inflammation and liver damage; this condition isreferred to as NASH. NASH is associated with an increased risk ofdeveloping liver cirrhosis and subsequence complications, includinghepatocellular carcinoma. A diagnosis of NAFLD or NASH can be made bymethods known in the art, e.g., by histological examination of liverbiopsy samples.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 A Functional Role of miR-128 in Lipid Homeostasis and InsulinSignaling

To determine whether miR-128 plays a role in lipid homeostasis andinsulin signaling, the following experiments were performed. First,expression of miR-128-1 was evaluated in 20 selected human tissues usingthe Taqman miRNA Assay Kit (Applied Biosystems). Total RNA samples werepurchased from Ambion, now an Applied Biosystems company. Briefly, 10 ngtotal RNA was reverse-transcribed using a miR-128-specific primer(comprising sequence complementary to part of SEQ NO:3), followed byquantitative real-time PCR with Taqman probes. Thereby, the U6spliceosomal RNA was used as an internal control. The results indicatedthat miR-128-1 was co-expressed with the host gene R3HDM1 in a number ofhuman tissues (FIGS. 7A-B). Both genes were shown to be expressed fairlyubiquitously. The function of R3HDM1 has not yet been determined. Theexpression profile of miR-128 shown in FIG. 8B reflects the combinedexpression of miR-128-1 (located in the R3HDM1 gene) and miR-128-2,whose genomic location is within the ARPP-21 host gene. miR-128-1 andmiR-128-2 have identical mature sequences, which are measured here.

To further determine whether miR-128 plays a role in regulating lipidhomeostasis and insulin signaling, experiments were performed to seewhat effect increased or decreased expression of miR-128 would have ongenes associated with lipid homeostasis, including LDLR, ABCA1, SIRT1,and CYP39A1, as well as the insulin-signaling component IRS1. Mutationsin LDLR cause the autosomal dominant disorder familialhypercholesterolemia (Brown and Goldstein, Scientific American. 1984,252:52-60; Leigh S E et al. Ann Hum Genet. 2008, 72:485-498). Withcholesterol as its substrate, the ABCA1 protein functions as acholesterol efflux pump in the cellular lipid removal pathway, therebypromoting HDL biosynthesis. Mutations in the ABCA1 gene have beenassociated with Tangier's disease, familial high-density lipoproteindeficiency, and elevated cardiovascular disease risk (Brooks-Wilson etal., (1999) Nature Genetics, 22:336-45; Hong et al., (2003)Atherosclerosis 164: 245-250). The CYP39A1 enzyme contributes to thecholesterol catabolic pathway in the liver, which converts cholesterolto bile acids, the primary mechanism for the removal of excesscholesterol from the body. Mutations in this CYP sub-family of enzymesare associated with defects in bile acid biosynthesis which caneventually lead to severe metabolic disorders (Wang et al., (2009) JLipid Res. 50:S406-411). SIRT1 regulates the function of many keytranscription factors in human metabolism. For example, it inhibitsSREBP activity, a master regulator of cholesterol and fat metabolism(Walker et al., (2010) Genes Dev. 24:1403-1417):

IRS1 plays a key role in transmitting signals from the insulin receptorto intracellular metabolic pathways. Mutations in the IRS1 gene areassociated with type II diabetes and susceptibility to insulinresistance (Berger et al. (2002), Diabet Med. 19:804-809; Eftychi Cetal. (2004) Diabetes: 53:870-873):

The effects of increasing expression of miR-128 were evaluated by theintroduction of excess miR-128-1 precursor oligonucleotides into humanHepG2 liver cells. The precursor oligonucleotides were double strandedand mimicked the precursor sequence of miR-128. The active strandcomprises the sequence ID NO.3. Antisense treated cells were harvested24 hours after transfection, whereas cells treated with Precursoroligonucleotides were harvested after 48 hours of transfection in wholecell extract buffer. Immunoblotting was carried out using proteinspecific antibodies according to manufacturer's protocol. Antibodies forLDLR, ABCA1 and CYP39A1 were from Abcam. SIRT1 antibody was from Cyclex,and the antibody for IRS1 from Cell Signaling.

As shown in FIGS. 8-11, increasing expression of miR-128 results indecreased expression of key cholesterol/lipid regulators such as LDLR,ABCA1, SIRT1, and CYP39A1, well as the insulin-signaling component IRS1.

Next, the effects of decreasing expression of miR-128 were evaluated bythe introduction of excess miR-128-1 antisense oligonucleotides intohuman HepG2 liver cells. The antisense oligos were purchased from Ambion(Applied Biosystems). Transfection studies were all carried out byelectroporation using cell-specific transfection reagents from LonzaCompany. Each transfection procedure contained 2×10⁶ HepG2 cells and 0.1nmol of antisense oligo. Upon transfection, cells were plated onpolylysine-coated plates and harvested for analysis 24 hours postincubation at 37° C. under 5% CO2. Cells were all grown in MEM mediumwith 10% FBS. The antisense sequence used in this study wasAAAGAGACCGGTTCACTGTGA (SEQ ID NO:7). According to the manufacturer theoligo contains a phosphorothioate backbone and 2′-Methoxy moieties.

Importantly, reducing miR-128 expression using antisenseoligonucleotides complementary to miR-128-1 cause increased expressionof LDLR, ABCA SIRT1 and IRS1, suggesting that antisense-basedtherapeutics could potentially impact the levels of these proteins inhuman liver (FIGS. 8-11).

These results support a central role of miR-128-1 in regulation ofCholesterol/lipid homeostasis and insulin signaling/energy homeostasis.

Example 2 miR-128 Regulates Hepatic LDLR Expression and LDL Uptake

To determine whether miR-128 can directly affect expression of LDLR, areporter construct was developed that included the LDLR-3′ UTR sequencelinked to a luciferase reporter. The construct contained the entire LDLR3′UTR sequence (2498 bp) downstream of the Luciferase gene (RenSP).Transfections were carried out using the LIPOFECTAMINE transfectionreagent. HEK293 cells were transfected with 10 ng of plasmid constructand 30 nM of Precursor miR-128 oligos, and the Luciferase actvity wasmeasured 24 hours post-transfection. A beta-Galactosidase expressionvector was cotransfected (100 ng) and its activity used as a normalizer.

When expressed in HEK 293 cells, the LDLR 3′UTR mediated strongrepression of the fused Luciferase reporter (FIG. 12A), and thisrepression was further increased upon introduction of additionalmiR-128-1 precursor oligonucleotides (FIG. 12B).

To determine whether miR-128 had any effects on lipid uptake, humanliver cells (HepG2) were treated with the antisense and precursor oligosas described above. The LDL uptake experiment was initiated by addingDil-LDL (Biomedical Technologies) in a final concentration of 10 μg/ml.The LDL uptake was carried out for 2 hours. Upon incubation, cells werewashed three times with cold PBS. Cells were finally lysed in SS buffercontaining 0.1 M NaOH and 0.1% SDS according to standard protocols(Stephan Z F & Yurachek E C, (1993) J Lipid Res 34: 325-330).

As in the HE293 cells, LDL uptake into human liver cells (HepG2) wasincreased upon treatment with miR-128-1 antisense oligonucleotides, anddecreased upon treatment with miR-128-1 precursor oligonucleotides(FIGS. 8A-B and 14A-C). These results clearly show that miR-128-1 hasfunctional effects on LDLR activity by post-transcriptional regulationof the expression of LDLR.

These data together provide critical support for the notion thatmiR-128-1 is an important regulator of hepatic LDLR expression and LDLuptake. Indeed, miR-128-1 is the first significant regulator of LDLRexpression since the discovery of the SREBP genes 17 years ago by Brownand Goldstein at UT Southwestern Medical Center in Dallas (Yokoyama etal., (1993) Cell, 75:187-195; Goldstein and Brown, (2009) ArteriosclerThromb Vasc Biol. 29:431-438).

Example 3 miR-128-1 Regulates Expression of the ATP-binding cassette A1(ABCA1) Cholesterol Transporter and SIRT1

The ABCA1 cholesterol transporter is another potential target ofmiR-128-1 (Table 1). ABCA1 is critical for the production of HDL by theliver, and also acts as a cholesterol efflux pump that extrudescholesterol and phospholipids to HDL from peripheral tissues and cells,including arterial macrophages, to promote reverse cholesterol transport(RCT) (Cuchel and Rader, Circulation 113 (21), 2548-2555 (2006)).Importantly, variations in the ABCA1 gene have been linked to impairedHDL synthesis and RCT, and may be associated with elevated risk foratherosclerosis (Rader et al., J Lipid Res 50 Suppl, S189-194 (2009)).

SIRT1 regulates the function of many key transcription factors in humanmetabolism. For example, it inhibits SREBP activity, a master regulatorof cholesterol and fat metabolism (Walker et al., (2010) Genes Dev.24:1403-1417).

To determine whether miR-128 can directly affect expression of ABCA1 andSIRT1, reporter constructs were developed that included either theABCA1-3′ UTR and SIRT1-3′ UTR sequence linked to a luciferase reporter.The constructs contained the SIRT1 or ABCA1 3′UTR sequences downstreamof the Luciferase gene (RenSP). Pre-plated HEK293 cells were transfectedwith 40 nM miR-128-1 precursor or precursor control (Ambion) in thepresence of 2 ng of ABCA1 or SIRT1 luciferase plasmid constructs,respectively, using lipofectamine 2000 (Invitrogen/Life Technologies).Cells were harvested 24-48 hours post-transfection and the luciferaseactivity measured following standard protocols (Promega). The resultsshow that miR-128-1 targets the ABCA1 and SIRT1 3′ UTRs forpost-transcriptional regulation. Introduction of human miR-128-1 causesa repression of of the Luciferase-ABCA1 (FIG. 13A) and SIRT1 (FIG. 13B)3′UTRs, showing specific targeting by miR-128-1 through their 3′UTR.

To further evaluate the effects of miR-128-1 ABCA1 expression, Huh-7cells were treated with 50 nM of Anti-miR-128-1 (Ambion) and plated athigh density in DMEM media containing 5% FBS. After 48 hrs cells wereharvested and protein amount was quantified by western blottinganalysis. As shown in FIG. 15, coordinated up regulation of ABCA1 andLDLR in Huh-7 human liver cells was seen in response to miR-128-1antisense inhibition. Thus, miR-128-1 indeed controls the levels ofABCA1 in human liver cells (FIG. 15).

Example 4 Effects of miR-128-1 on Human Liver Cell Whole GenomeExpression

MicroRNAs typically regulate many targets, frequently by controllingmRNA stability⁴⁸. TargetScan provides a mathematical predictor ofmicroRNA targets. To explore the reach of miR-128-1 in cellsempirically, DNA microarray analysis was employed to obtain acomprehensive list of potential miR-128-1 targets. These studiesidentify novel pathways regulated by miR-128-1 that may affecttherapeutic targeting efforts. Samples were analyzed on Affymetrix humangenome U133 Plus 2.0 arrays by Affymetrix readers. Statistical andbioinformatics analyses (e.g., Gene Ontology (GO) analysis (Osborne etal., Methods Mol Biol 377, 223-242 (2007)) and Ingenuity PathwayAnalysis (Ganter and Giroux, Curr Opin Drug Discov Devel 11 (1), 86-94(2008); Thomas and Bonchev, Hum Genomics 4 (5), 353-360 (2010))) wereperformed. Expression changes of selected genes of interest wereconfirmed by qRT-PCR.

DNA microarray analysis of HepG2 cells transfected with miR-128-1precursor oligonucleotides was performed. Unsupervised hierarchicalclustering analysis revealed that several predicted and verified targetsof miR-128-1, including LDLR and ABCA1, were indeed down-regulated asexpected in miR-128-1-treated cell as compared with cells transfectedwith control oligonucleotides (FIG. 16). Unexpectedly, there wasdecreased expression of several additional genes involved incholesterol/lipid trafficking, but which were not predicted as targetsof miR-128-1, including ApoE, ApoA1, and several genes in the ApoCcluster (FIG. 16). Conversely, ApoB was found to be upregulated HepG2cells with increased miR-128-1 levels. These initial findings suggestthat miR-128-1 may coordinately control both directly and indirectly theproduction of key lipoproteins involved in cholesterol/lipidtrafficking, with important relevance to cardiovascular biology. Indeed,ApoB is associated with pro-atherogenic LDL, whereas ApoA1 is associatedwith anti-atherogenic HDL (Walldius and Jungner, J. Intern Med 259 (5),493-519 (2006)). In accord with this notion, unbiased Gene Ontologyanalysis of the entire DNA microarray dataset by DAVID revealed a highlystatistically significant association with the “Cardiovascular Disease”and “Atherosclerosis” GO terms/gene sets.

Example 5 Functional Contribution of miR-128-1 to Control ofCholesterol/Lipid and Energy Homeostasis in Vivo

It was hypothesized that miR-128-1 manipulation will affectlipid/cholesterol storage and energy usage in mice, and that the effectsof miR-128-1 overexpression will be exacerbated by a high-fat diet whileantisense treatment will provide protection from deleterious effectsnormally associated with this diet. In two independent experiments,C57BL/6J mice (n=10) were placed on a high-fat diet were treated with alentivirus encoding for human miR-128-1 precursor obtained from SystemBiosciences Inc. (SBI). The modified pMIRNA1 consists of the nativehuman miR-128-1 stem loop structure and 200-400 base pairs of upstreamand downstream flanking genomic sequence (see FIG. 18, SEQ ID NO:8).Mice were injected with 2-5×10⁷ IFU/mouse of the lentiviral construct in300 μl PBS via either retro-orbital or tail-vein injections. At days 9(Exp. 1) and 14 (Exp. 2) after the injection, the mice were sacrificedand total blood was collected. The cholesterol distribution in plasmalipoproteins fractions was assessed by fast-performance liquidchromatography (FPLC) gel filtration using 250 μl of pooled serum.

The mice overexpressing miR-128-1 exhibited several striking phenotypes.First, while these mice ate much less food (data not shown), theirweight was very similar to controls (29.46+/−0.45 (s.e.m) grams{miR-128-1} vs. 30.59+/−0.47 (s.e.m.) grams {control}, p=0.19, n=10 pergroup). This suggests that miR-128-1 indeed can increase the ability ofan animal to maintain weight on a reduced number of calories, as thestudies associating its locus with feed efficiency in cattle suggested.Second, serum profiling showed that LDL-cholesterol was raised andHDL-cholesterol lowered compared to control virus-injected mice (FIGS.17A-B; pooled serum from 10 mice for each group). Thus the hypothesisregarding miR-128-1 effects on cholesterol trafficking has beenvalidated in vivo. Third, visual inspection revealed that miR-128-1overexpressing mice exhibited an unexpected and distinctive “greasy fur”phenotype. This initial experiment supports the feasibility of ourapproach and suggests that miR-128-1 overexpression may cause alteredanimal metabolism, possibly related to lipid homeostasis/fat storage.

Example 6 Effect of miR-128-1 Manipulations on Metabolic Homeostasis inMice

These studies are expanded to include analyses of mice on differenttypes of diets either overexpressing miR-128-1, or subjected tomiR-128-1 antisense inhibition. First, twenty C57BL/6J mice on regularchow are subjected to tail-vein injection with a miR-128-1 lentivirus,e.g., as described above, while another twenty mice are injected withcontrol (empty vector) virus. To determine the potential collaborationof elevated miR-128-1 expression with high-fat diets, similar in vivoexperiments are performed with animals fed either a Western-type diet(41% of calories from fat), or a high-fat diet (60% of calories as fat).

The animals are weighed every three days. Food intake, and generalhealth and activity, will be monitored daily. After 10 days, blood willbe drawn and serum levels of total cholesterol, triglycerides, glucose,and liver enzymes (ALT/AST, for toxicity) will be analyzed. In addition,FPLC analysis of serum lipoproteins (LDL, HDL, and VLDL) will be carriedout.

Next, the effects of miR-128-1 overexpression are “rescued” by anantisense approach, and the effects of inhibition of endogenousmiR-128-1 are assessed. The twenty mice from each cohort above aredivided into two groups, one receiving subcutaneous injection with 5mg/kg of LNA-antisense oligonucleotides complementary to miR-128-1(Exigon), while the other 10 mice receive injection with PBS (control).This LNA-antisense treatment regimen is similar to that we employed in aprevious in vivo study of miR-33 (Nafaji-Shoushtari et al., Science 328(5985), 1566-1569 (2010)). After one week, the mice are sacrificed andserum is collected, as well as liver and adipose tissues, for analysisas previously described.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of reducing obesity, treating orreducing predisposition to insulin resistance, and/or treating orreducing predisposition to type II diabetes, in a subject, the methodcomprising administering to the subject an inhibitory nucleic acidsequence that is complementary to all or part of any of SEQ ID NOs:1-6in an amount sufficient to decrease obesity, treat or reducepredisposition to insulin resistance, and/or to treat or reducepredisposition to type II diabetes, thereby decreasing obesity, treatingor reducing predisposition to insulin resistance, and/or treating orreducing predisposition to type II diabetes, in the subject.
 2. Themethod of claim 1, wherein the inhibitory nucleic acid is complementaryto all or part of SEQ ID NO:2.
 3. The method of claim 1, wherein theinhibitory nucleic acid is complementary to at least nucleotides 2-7(5′-CACAGU-3′) of SEQ ID NO:3.
 4. The method of claim 1, wherein theinhibitory nucleic acid is an antisense oligonucleotide.
 5. The methodof claim 4, wherein the antisense oligonucleotide comprises a sequencethat is complementary to SEQ ID NO:3.
 6. The method of claim 4, whereinthe antisense oligonucleotide is an antagomir.
 7. The method of claim 1,wherein the inhibitory nucleic acid is an interfering RNA.
 8. The methodof claim 7, wherein the interfering RNA is a small hairpin RNA (shRNA)or small interfering RNA (siRNA).
 9. The method of claim 1, wherein theinhibitory nucleic acid sequence inhibits post-transcriptionalprocessing of SEQ ID NO:1 or
 5. 10. The method of claim 1, wherein thesubject has metabolic syndrome or Type 2 diabetes.
 11. The method ofclaim 10, further comprising selecting a subject on the basis that theyhave metabolic syndrome or Type 2 diabetes.
 12. The method of claim 1,further comprising detecting the presence of one or more allelesassociated with increased levels of miR-128 and/or predisposition toincreased levels of serum lipids, and optionally selecting a subject onthe basis of the presence of an allele associated with increased levelsof miR-128.
 13. The method of claim 1, further comprising selecting asubject who has a BMI of 25 or above.
 14. The method of claim 1, whereinthe inhibitory nucleic acid has at least one locked nucleotide.
 15. Themethod of claim 1, wherein the inhibitory nucleic acid has aphosphorothioate backbone.
 16. The method of claim 1, wherein theinhibitory nucleic acid decreases obesity in the subject.
 17. The methodof claim 1, wherein the inhibitory nucleic acid treats or reducespredisposition to insulin resistance in the subject.
 18. The method ofclaim 1, wherein the inhibitory nucleic acid treats or reducespredisposition to type II diabetes in the subject.
 19. The method ofclaim 1, wherein the inhibitory nucleic acid increases adiposeexpression of Sirtuin 1 (SIRT1).
 20. The method of claim 1, wherein theinhibitory nucleic acid increases expression of Insulin receptorsubstrate 1 (IRS1).